Our long term goal is to develop a safe, effective vaccine to prevent respiratory syncytial virus (RSV) disease. RSV is the leading cause of bronchiolitis, viral pneumonia, respiratory failure, and mechanical ventilation in infants. It causes >120,000 infant hospitalizations per year in the USA and is the leading cause of infant viral death. In the elderly, RSV causes one third of pneumonia hospitalizations. In children and adults, RSV infection is a major cause of acute asthma exacerbations. There is no licensed vaccine. Experimental RSV vaccines including inactivated, live attenuated, subunit, viral-vectored, and DNA have been developed and tested in animal models and small clinical trials. However, these have been largely unsuccessful. Inactivated and subunit vaccines have a history of causing immunopathology. Live attenuated RSV vaccines are safe. However, attenuated RSVs characteristically induce transient/poor immunity like wild-type RSV. We are developing novel virus-like particle (VLP) RSV vaccines. VLPs are genetically engineered complexes of proteins in a particulate virus-like structure that lacks viral genetic material and therefore cannot replicate. Viral proteins presented as VLPs are highly immunogenic and induce protective humoral, cellular, and mucosal immune responses. We have extensive experience expressing viral proteins on VLPs and evaluating VLP-induced immunity. The RSV fusion (F) and attachment glycoprotein (G) contain all known neutralizing antibody epitopes and several T cell epitopes. We hypothesize that VLPs expressing RSV F, G, or both F and G surface will induce strong RSV-specific immune responses and immunity. In Aim 1, we will generate VLPs using a recombinant baculovirus system to express VLP proteins in insect cells and gradient ultracentrifugation to purify VLPs. We have experience with these methods which are FDA approved for human use and scalable for production. We will generate RSV-G, RSV-F, and RSV-GF VLPs by co-expressing these proteins with influenza matrix M1 protein as the VLP core. VLPs co-expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) proteins exhibit enhanced cellular and humoral responses. We will generate RSV VLPs with and without anchored GM-CSF. Also, we will optimize immunogenicity by testing three different co-administered adjuvants, aluminum hydroxide (alum), CpG oligodeoxynucleotides (CpG ODN), and monophosphoryl lipid (MPL). The latter two adjuvants stimulate toll- like receptor signaling pathways known to be important for anti-RSV responses. In Aim 2, we will define the immunogenicity and efficacy of these RSV VLP vaccines in a mouse model. Mice will be primed and boosted with VLPs intranasally. RSV-specific antibody (IgG and IgA) and T cell responses will be quantified after immunization. To determine protection, we will challenge mice with a recombinant, chimeric RSV strain known to give higher lung viral loads than laboratory RSV strain in BALB/c mice. Collectively, these experiments will advance RSV VLP vaccine development and may lead to a much needed approved RSV vaccine. PUBLIC HEALTH RELEVANCE: Respiratory syncytial virus (RSV) causes apprimately 120,000 infant hospitalizations in the US each year and is the leading cause of bronchiolitis and viral death in infants. Despite decades of research with traditional vaccine approaches, there are no approved RSV vaccines. We are advancing virus-like particle (VLP) vaccines for RSV, and we predict RSV VLPs will be effective and safe vaccines that prevent RSV disease.